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Photocaged, activity‐based ubiquitin (Ub) probes (Ub‐ABPs) have been developed for the time‐resolved probing of deubiquitinating enzyme (DUB) activities, but many Ub‐ABPs are still challenging to photocage because their warheads (e.g. propargylamide (PA) or dehydroalanine (Dha)) are difficult to temporally block and activate. Here, we describe a new C‐terminal backbone modification strategy for the construction of photocaged Ub‐ABPs in which a light‐sensitive group is placed at the backbone amide bond of the Ub Gly75. This strategy enabled the facile generation of cell‐permeable photocaged Ub‐PA and Dha probes that could be activated to capture DUBs after photo‐irradiation, and were used to profile DUBs in cells under specially designed conditions (e.g. in cells experiencing oxidative stress) or DUBs with isopeptide linkage selectivity. This backbone modification strategy is anticipated to provide a general solution for the development of photocaged Ub ABPs bearing any warheads for DUB profiling. [ABSTRACT FROM AUTHOR]

Activity‐based E2 conjugating enzyme (E2)‐ubiquitin (Ub) probes have recently emerged as effective tools for studying the molecular mechanism of E3 ligase (E3)‐catalyzed ubiquitination. However, the preparation of existing activity‐based E2‐Ub probes depends on recombination technology and bioconjugation chemistry, limiting their structural diversity. Herein we describe an expedient total chemical synthesis of an E2 enzyme variant through a hydrazide‐based native chemical ligation, which enabled the construction of a structurally new activity‐based E2‐Ub probe to covalently capture the catalytic site of Cys‐dependent E3s. Chemical cross‐linking coupled with mass spectrometry (CXMS) demonstrated the utility of this new probe in structural analysis of the intermediates formed during Nedd4 and Parkin‐mediated transthiolation. This study exemplifies the utility of chemical protein synthesis for the development of protein probes for biological studies. [ABSTRACT FROM AUTHOR]